Optical flow cytometry is an established means of counting and classifying particles contained within a fluidic sample (Shapiro, H. M., Practical flow cytometry, Third Edition, Wiley-Liss publishers, New York, 1995). One application involves the analysis of a blood sample for the purposes of determining the numbers of platelets, red blood cells (RBCs), and white blood cells (WBCs) per unit volume. This is a common clinical measurement, and optical cytometers have been incorporated into a number of commercial hematology analyzers. Recently, microfluidic techniques have been employed for the purposes of developing cytometers which require smaller sample and reagent volumes (Altendorf, E. et al., "Differential blood cell counts obtained using a microchannel based flow cytometer," Sensors and Actuators [1997] 1:531-534; Sobek, D. et al., "Microfabricated fused silica flow chambers for flow cytometry," Solid-State Sensor and Actuator Workshop, Hilton Head Island, S.C. [1994]; Miyake, R. et al., "A development of micro sheath flow chamber," Proceedings of the IEEE micro electro mechanical systems workshop, Nara, Japan [1991] 265-270). Analytical instruments based on these efforts will be smaller and more portable than conventional devices.
Knowledge of the number and nature of blood cells is important for disease diagnosis. For this reason, complete blood counts and white cell differentials are common clinical diagnostic tests carried out using a hematology analyzer. Forward angle light scattering (FALS) is sensitive to particle size and can be used to distinguish platelets from red blood cells. Small angle scattering (SALS) or large angle light scattering (LALS) in combination with FALS can distinguish within the WBCs between granulocytes, lymphocytes and monocytes (Salzman, G. C. et al. (1975), "Cell Classification by Laser Light Scattering: Identification and Separation of Unstained Leukocytes," Acta Cytologica 19:374-377). However, within the granulocytes, SALS and FALS cannot clearly distinguish between eosinophils and the remaining granulocytes such as neutrophils and basophils.
The difference in intensity of scattered light between s-polarized and p-polarized light can be used to further distinguish between the granulocytes. At small angles scattering of the two polarizations is indistinguishable. At large angles, WBC sized structures with no internal structure show only a small difference between the scattered light intensity of the two polarizations. Granulocyte WBCs, having an internal structure comprising numerous small granules, exhibit a difference in scattering intensity between the polarizations. In eosinophils the granules are birefringent and act to depolarize the scattered light, thereby reducing the difference in scattering intensity between the two polarizations. This depolarization has been used to distinguish cell types (Terstappen, L.W.M.M. et al. (1988), "Four-Parameter White Blood Cell Differential Counting Based on Light Scattering Measurements," Cytometry 9:39-43; de Grooth et al., U.S. Pat. No. 5,017,497; Marshall, U.S. Pat. No. 5,510,267. The depolarization was measured by impinging polarized light on a sample, collecting the large angle scattered light at a single large angle, splitting the collected light into two beams and measuring the scattered light in the two beams using two detectors, one for orthogonal light scattering of all polarizations, and the second preceded by a polarizing filter to measure depolarized orthogonal light scattering.